RESUMO
Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.
Assuntos
COVID-19/transmissão , COVID-19/virologia , Mutação , SARS-CoV-2/classificação , SARS-CoV-2/fisiologia , Replicação Viral , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Animais de Laboratório/virologia , COVID-19/veterinária , Cricetinae , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Furões/virologia , Humanos , Masculino , Mesocricetus/virologia , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , SARS-CoV-2/crescimento & desenvolvimento , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Virulência/genéticaRESUMO
During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.
Assuntos
COVID-19/transmissão , COVID-19/virologia , Mutação , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral/genética , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Brônquios/citologia , Brônquios/virologia , COVID-19/epidemiologia , Linhagem Celular , Células Cultivadas , Cricetinae , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Furões/virologia , Efeito Fundador , Técnicas de Introdução de Genes , Aptidão Genética , Humanos , Masculino , Mesocricetus , Camundongos , Mucosa Nasal/citologia , Mucosa Nasal/virologia , Ligação Proteica , RNA Viral/análise , Receptores de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidadeRESUMO
African Swine Fever virus (ASFV) is a large double-enveloped DNA virus of the Asfarviridae family that causes a lethal hemorrhagic disease in domestic pigs and wild boars. Since 2007, a highly virulent genotype II strain has emerged and spread in Europe and South-East Asia, where millions of animals succumbed to the disease. Field- and laboratory-attenuated strains of ASFV cause highly variable clinical disease severity and survival, and mechanisms remain unclear. We hypothesized that the immunological and hygienic status of pigs is a determinant of ASF disease course. Here we compared the immunological profile at baseline and in response to ASFV infection in specific pathogen-free (SPF) and farm-raised Large White domestic pigs. At steady state, SPF pigs showed lower white blood cell counts and a lower basal inflammatory and antiviral transcriptomic profile compared to farm pigs, associated with profound differences in gut microbiome composition. After inoculation with a highly virulent ASFV genotype II strain (Armenia 2008), severe clinical signs, viremia and pro-inflammatory cytokines appeared sooner in SPF pigs, indicating a reduced capacity to control early virus replication. In contrast, during infection with an attenuated field isolate (Estonia 2014), SPF pigs presented a milder and shorter clinical disease with full recovery, whereas farm pigs presented severe protracted disease with 50% lethality. Interestingly, farm pigs showed higher production of inflammatory cytokines, whereas SPF pigs produced more anti-inflammatory IL-1ra early after infection and presented a stronger expansion of leukocytes in the recovery phase. Altogether, our data indicate that the hygiene-dependent innate immune status has a double-edge sword impact on immune responses in ASF pathogenesis. While the higher baseline innate immune activity helps the host in reducing initial virus replication, it promotes immunopathological cytokine responses, and delays lymphocyte proliferation after infection with an attenuated strain. Such effects should be considered for live vaccine development and vigilance.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Vírus da Febre Suína Africana/genética , Animais , Citocinas , Higiene , Índice de Gravidade de Doença , Sus scrofa , SuínosRESUMO
The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4+ and CD8+ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity.
Assuntos
Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções Respiratórias/imunologia , Linfócitos T/patologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/imunologia , Células Cultivadas , Pré-Escolar , Modelos Animais de Doenças , Progressão da Doença , Humanos , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Pulmão/virologia , Infecções por Vírus Respiratório Sincicial/congênito , Infecções por Vírus Respiratório Sincicial/patologia , Infecções Respiratórias/congênito , Infecções Respiratórias/patologia , Ovinos/crescimento & desenvolvimento , Ovinos/imunologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Eosinophils are white blood cells that contribute to the regulation of immunity and are involved in the pathogenesis of numerous inflammatory diseases. In contrast to other cells of the immune system, no information is available regarding the role of autophagy in eosinophil differentiation and functions. To study the autophagic pathway in eosinophils, we generated conditional knockout mice in which Atg5 is deleted within the eosinophil lineage only (designated Atg5eoΔ mice). Eosinophilia was provoked by crossbreeding Atg5eoΔ mice with Il5 (IL-5) overexpressing transgenic mice (designated Atg5eoΔIl5tg mice). Deletion of Atg5 in eosinophils resulted in a dramatic reduction in the number of mature eosinophils in blood and an increase of immature eosinophils in the bone marrow. Atg5-knockout eosinophil precursors exhibited reduced proliferation under both in vitro and in vivo conditions but no increased cell death. Moreover, reduced differentiation of eosinophils in the absence of Atg5 was also observed in mouse and human models of chronic eosinophilic leukemia. Atg5-knockout blood eosinophils exhibited augmented levels of degranulation and bacterial killing in vitro. Moreover, in an experimental in vivo model, we observed that Atg5eoΔ mice achieve better clearance of the local and systemic bacterial infection with Citrobacter rodentium. Evidence for increased degranulation of ATG5low-expressing human eosinophils was also obtained in both tissues and blood. Taken together, mouse and human eosinophil hematopoiesis and effector functions are regulated by ATG5, which controls the amplitude of overall antibacterial eosinophil immune responses.
Assuntos
Proteína 5 Relacionada à Autofagia/fisiologia , Eosinófilos/fisiologia , Mielopoese/fisiologia , Animais , Proteína 5 Relacionada à Autofagia/biossíntese , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Medula Óssea/patologia , Sistemas CRISPR-Cas , Degranulação Celular , Linhagem Celular Tumoral , Células Cultivadas , Citrobacter rodentium , Ensaio de Unidades Formadoras de Colônias , Infecções por Enterobacteriaceae/imunologia , Eosinófilos/citologia , Eosinófilos/imunologia , Humanos , Síndrome Hipereosinofílica/sangue , Síndrome Hipereosinofílica/patologia , Interleucina-5/genética , Contagem de Leucócitos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Neutrophil extracellular trap (NET) formation is a cellular function of neutrophils that facilitates the immobilization and killing of invading microorganisms in the extracellular milieu. To form NETs, neutrophils release a DNA scaffold consisting of mitochondrial DNA binding granule proteins. This process does not depend on cell death, but requires glycolytic ATP production for rearrangements in the microtubule network and F-actin. Such cytoskeletal rearrangements are essential for both mitochondrial DNA release and degranulation. However, the formation of NETs has also been described as a distinct form of programed, necrotic cell death, a process designated "NETosis." Necrotic cell death of neutrophils is associated with the permeabilization of both plasma and nuclear membranes resulting in a kind of extracellular cloud of nuclear DNA. The molecular mechanisms eliciting necrotic neutrophil death have been investigated and appear to be different from those responsible for NET formation following mitochondrial DNA release. Here, we discriminate between the mechanisms responsible for the release of mitochondrial versus nuclear DNA and address their respective functions. Our aim is to clarify existing differences of opinion in the fields of NET formation and neutrophil death.
Assuntos
Armadilhas Extracelulares/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Morte Celular , Humanos , NecroseRESUMO
Proteinase 3 (PR3) is a myeloid serine protease expressed in neutrophils, monocytes, and macrophages. PR3 has a number of well-characterized proinflammatory functions, including cleaving and activating chemokines and controlling cell survival and proliferation. When presented on the surface of apoptotic neutrophils, PR3 can disrupt the normal anti-inflammatory reprogramming of macrophages following the phagocytosis of apoptotic cells. To better understand the function of PR3 in vivo, we generated a human PR3 transgenic mouse (hPR3Tg). During zymosan-induced peritonitis, hPR3Tg displayed an increased accumulation of neutrophils within the peritoneal cavity compared with wild-type control mice, with no difference in the recruitment of macrophages or B or T lymphocytes. Mice were also subjected to cecum ligation and puncture, a model used to induce peritoneal inflammation through infection. hPR3Tg displayed decreased survival rates in acute sepsis, associated with increased neutrophil extravasation. The decreased survival and increased neutrophil accumulation were associated with the cleavage of annexin A1, a powerful anti-inflammatory protein known to facilitate the resolution of inflammation. Additionally, neutrophils from hPR3Tg displayed enhanced survival during apoptosis compared with controls, and this may also contribute to the increased accumulation observed during the later stages of inflammation. Taken together, our data suggest that human PR3 plays a proinflammatory role during acute inflammatory responses by affecting neutrophil accumulation, survival, and the resolution of inflammation.
Assuntos
Mieloblastina/metabolismo , Neutrófilos/imunologia , Cavidade Peritoneal/patologia , Peritonite/imunologia , Sepse/imunologia , Animais , Anexina A1/metabolismo , Apoptose , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mieloblastina/genética , Peritonite/induzido quimicamente , Fagocitose , Sepse/induzido quimicamente , ZimosanRESUMO
Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followed by a protracted disease course in SJL/J mice. In contrast, cigarette smoke exposure ameliorated the EAE clinical score in C57BL/6J mice. Our exploratory studies therefore show that genetic background influences the effects of cigarette smoke on autoimmune neuroinflammation. Importantly, our findings expose the challenge of identifying an animal model for studying the influence of cigarette smoke in MS.
Assuntos
Encefalomielite Autoimune Experimental/diagnóstico , Encefalomielite Autoimune Experimental/etiologia , Patrimônio Genético , Fumar/efeitos adversos , Idade de Início , Animais , Biópsia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/metabolismo , Imuno-Histoquímica , Camundongos , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/etiologia , Esclerose Múltipla/metabolismo , Fenótipo , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Medula Espinal/metabolismo , Medula Espinal/patologiaAssuntos
Vacinas contra COVID-19 , COVID-19 , Feminino , Masculino , Humanos , SARS-CoV-2 , COVID-19/prevenção & controle , Vacinação , ImunidadeRESUMO
Neutrophils constitute a crucial component of the innate immune defenses against microbes. Produced in the bone marrow and patrolling in blood vessels, neutrophils are recruited to injured tissues and are immediately active to contain pathogen invasion. Neutrophils undergo programmed cell death by multiple, context-specific pathways, which have consequences on immunopathology and disease outcome. Studies in the last decade indicate additional functions for neutrophils - or a subset of neutrophils - in modulating adaptive responses and tumor progression. Neutrophil granules contain abundant amounts of various proteases, which are directly implicated in protective and pathogenic functions of neutrophils. It now emerges that neutral serine proteases such as cathepsin G and proteinase-3 also contribute to the neutrophil life cycle, but do so via different pathways than that of the aspartate protease cathepsin D and that of mutants of the serine protease elastase. The aim of this review is to appraise the present knowledge of the function of neutrophil granule proteases and their inhibitors in neutrophil cell death, and to integrate these findings in the current understandings of neutrophil life cycle and programmed cell death pathways.
Assuntos
Neutrófilos/citologia , Neutrófilos/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Grânulos Citoplasmáticos/enzimologia , Retroalimentação Fisiológica , Humanos , Elastase de Leucócito/genética , Mutação , Mielopoese/fisiologia , Neutropenia/etiologia , Neutrófilos/imunologia , Serpinas/metabolismoRESUMO
Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.
Assuntos
Movimento Celular/imunologia , Armadilhas Extracelulares/imunologia , Infecções/imunologia , Neutrófilos/imunologia , Animais , Sobrevivência Celular/imunologia , Humanos , Infecções/patologia , Inflamação/imunologia , Inflamação/patologia , Neutrófilos/patologiaRESUMO
CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the ß-sheet also maintained â¼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.
Assuntos
Anti-Infecciosos/farmacologia , Quimiocinas CXC/farmacologia , Infecções Pneumocócicas/imunologia , Infecções Respiratórias/imunologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/imunologia , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocinas CXC/química , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Quimiotaxia/efeitos dos fármacos , DNA Bacteriano , Modelos Animais de Doenças , Interleucina-8/farmacologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mieloblastina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Permeabilidade/efeitos dos fármacos , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/genética , Streptococcus pneumoniae/ultraestruturaRESUMO
Pneumonia is a leading cause of hospitalization in patients with chronic obstructive pulmonary disease (COPD). Although most patients with COPD are smokers, the effects of cigarette smoke exposure on clearance of lung bacterial pathogens and on immune and inflammatory responses are incompletely defined. Here, clearance of Streptococcus pneumoniae and Pseudomonas aeruginosa and associated immune responses were examined in mice exposed to cigarette smoke or after smoking cessation. Mice exposed to cigarette smoke for 6 weeks or 4 months demonstrated decreased lung bacterial burden compared with air-exposed mice when infected 16 to 24 hours after exposure. When infection was performed after smoke cessation, bacterial clearance kinetics of mice previously exposed to smoke reversed to levels comparable to those of control mice, suggesting that the observed defects were not dependent on adaptive immunological memory to bacterial determinants found in smoke. Comparing cytokine levels and myeloid cell production before infection in mice exposed to cigarette smoke with mice never exposed or after smoke cessation revealed that reduced bacterial burden was most strongly associated with higher levels of IL-1ß and granulocyte-macrophage colony-stimulating factor in the lungs and with increased neutrophil reserve and monocyte turnover in the bone marrow. Using Serpinb1a-deficient mice with reduced neutrophil numbers and treatment with granulocyte colony-stimulating factor showed that increased neutrophil numbers contribute only in part to the effect of smoke on infection. Our findings indicate that cigarette smoke induces a temporary and reversible increase in clearance of lung pathogens, which correlates with local inflammation and increased myeloid cell output from the bone marrow.
Assuntos
Imunidade Inata , Pulmão/imunologia , Células Mieloides/imunologia , Pneumonia Pneumocócica/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Fumar/imunologia , Streptococcus pneumoniae/imunologia , Animais , Carga Bacteriana , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interações Hospedeiro-Patógeno , Mediadores da Inflamação/imunologia , Interleucina-1beta/imunologia , Cinética , Labirintite/imunologia , Labirintite/microbiologia , Pulmão/microbiologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/microbiologia , Células Mieloides/microbiologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Pneumonia Pneumocócica/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/metabolismo , Serpinas/genética , Serpinas/metabolismo , Fumar/efeitos adversos , Streptococcus pneumoniae/patogenicidadeRESUMO
Serpinb1 is an inhibitor of neutrophil granule serine proteases cathepsin G, proteinase-3 and elastase. One of its core physiological functions is to protect neutrophils from granule protease-mediated cell death. Mice lacking Serpinb1a (Sb1a-/-), its mouse ortholog, have reduced bone marrow neutrophil numbers due to cell death mediated by cathepsin G and the mice show increased susceptibility to lung infections. Here, we show that conditional deletion of Serpinb1a using the Lyz2-cre and Cebpa-cre knock-in mice effectively leads to recombination-mediated deletion in neutrophils but protein-null neutrophils were only obtained using the latter recombinase-expressing strain. Absence of Serpinb1a protein in neutrophils caused neutropenia and increased granule permeabilization-induced cell death. We then generated transgenic mice expressing human Serpinb1 in neutrophils under the human MRP8 (S100A8) promoter. Serpinb1a expression levels in founder lines correlated positively with increased neutrophil survival when crossed with Sb1a-/- mice, which had their defective neutrophil phenotype rescued in the higher expressing transgenic line. Using new conditional and transgenic mouse models, our study demonstrates the presence of a relatively low Serpinb1a protein threshold in neutrophils that is required for sustained survival. These models will also be helpful in delineating recently described functions of Serpinb1 in metabolism and cancer.
Assuntos
Deleção de Genes , Células Mieloides/metabolismo , Neutrófilos/citologia , Serpinas/deficiência , Serpinas/genética , Alelos , Animais , Sobrevivência Celular , Técnicas de Introdução de Genes , Humanos , Camundongos Transgênicos , Recombinação GenéticaRESUMO
Neutrophils are essential to combat infectious agents but contribute to collateral inflammatory damage. Likewise, neutrophils can kill cancer cells and have been shown to promote malignant growth and metastasis through immunosuppressive functions. Two articles in a recent issue of Nature reveal new mechanisms by which tumors induce changes in neutrophil phenotype through production of inflammatory cytokines. Although the two studies report different outcomes on the effects of neutrophils on tumor growth and metastasis, they delineate novel molecular pathways influencing neutrophil phenotype that may provide new approaches to harnessing neutrophil functions in the treatment of cancer.
Assuntos
Neoplasias da Mama/imunologia , Neutrófilos/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Citocinas/fisiologia , Progressão da Doença , Feminino , Humanos , Terapia de Imunossupressão , Infiltração de Neutrófilos , FenótipoRESUMO
Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1(-/-)) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia of sB1(-/-) mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1(-/-) mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1(-/-) PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent l-leucyl-l-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity.
Assuntos
Catepsina G/antagonistas & inibidores , Neutrófilos/fisiologia , Serpinas/fisiologia , Animais , Catepsina G/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Regulação para Baixo/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Especificidade de Órgãos/genética , Serpinas/genética , Serpinas/metabolismoRESUMO
Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Animais , Camundongos , SARS-CoV-2/metabolismo , Neutrófilos/metabolismo , Glicoproteína da Espícula de Coronavírus , Inflamação , Serina Proteases/metabolismoRESUMO
Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including 'one-to-stop' (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Genoma Viral , SARS-CoV-2 , Vacinas Atenuadas , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/administração & dosagem , SARS-CoV-2/genética , SARS-CoV-2/imunologia , COVID-19/prevenção & controle , COVID-19/transmissão , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Animais , Genoma Viral/genética , Humanos , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Feminino , Chlorocebus aethiops , Modelos Animais de Doenças , Células Vero , Anticorpos Neutralizantes/imunologiaRESUMO
Chronic obstructive pulmonary disease (COPD) is characterized by emphysema and chronic bronchitis and is a leading cause of morbidity and mortality worldwide. Tobacco smoke and deficiency in α1-antitrypsin (AAT) are the most prominent environmental and genetic risk factors, respectively. Yet the pathogenesis of COPD is not completely elucidated. Disease progression appears to include a vicious circle driven by self-perpetuating lung inflammation, endothelial and epithelial cell death, and proteolytic degradation of extracellular matrix proteins. Like AAT, serpinB1 is a potent inhibitor of serine proteases including neutrophil elastase and cathepsin G. Because serpinB1 is expressed in myeloid and lung epithelial cells and is protective during lung infections, we investigated the role of serpinB1 in preventing age-related and cigarette smoke-induced emphysema in mice. Fifteen-month-old mice showed increased lung volume and decreased pulmonary function compared with young adult mice (3 mo old), but no differences were observed between serpinB1-deficient (KO) and wild-type (WT) mice. Chronic exposure to secondhand cigarette smoke resulted in structural emphysematous changes compared with respective control mice, but no difference in lung morphometry was observed between genotypes. Of note, the different pattern of stereological changes induced by age and cigarette smoke suggest distinct mechanisms leading to increased airway volume. Finally, expression of intracellular and extracellular protease inhibitors were differently regulated in lungs of WT and KO mice following smoke exposure; however, activity of proteases was not significantly altered. In conclusion, we showed that, although AAT and serpinB1 are similarly potent inhibitors of neutrophil proteases, serpinB1 deficiency is not associated with more severe emphysema.
Assuntos
Enfisema Pulmonar/genética , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Serpinas/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Mutação/genética , Pneumonia/induzido quimicamente , Pneumonia/genética , Pneumonia/patologia , Serpinas/deficiência , Fumaça/efeitos adversosRESUMO
Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1(-/-) mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein-D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance in serpinb1(-/-) mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection.